Autism spectrum disorder is currently defined as a single neurodevelopmental disorder characterized by impairments in social communication and the presence of restricted interests and repetitive behaviors. However, there is mounting evidence that autism spectrum disorder represents a collection of overlapping neurodevelopmental disorders, resulting from a variety of neuroanatomical, neurophysiological, neuroimmunologic and/or genetic abnormalities. This research program aims to characterize the behavioral and biological manifestations of these differing presentations of ASD and to identify their unique features in order to facilitate development of effective therapeutic and preventive interventions. During the reporting period, this project analyzed data from a longitudinal, phenotyping investigation of 106 young children (ages 1-6 years) with autism, 60 age and sex-matched typically developing controls and a group of 25 children with non-autistic developmental delays (Protocol NCT 00298246). The focus of analyses now is on the longitudinal data. Analyses included comprehensive medical and developmental histories; neuropsychological, medical and neurological evaluations; assays of blood, urine and cerebrospinal fluid (CSF) samples; and also a variety of specialized studies, including routine and overnight electroencephalograms (EEGs); modified polysomnography to evaluate sleep architecture; magnetic resonance imaging (MRI scans); genetic assays; and dysmorphology evaluations. Analyses of behavioral data included a recent investigation of specific nonverbal communicative behaviors, finding gesture use under the age of 4 in this sample predicted receptive language outcomes. We recently explored CSF and blood samples in this cohort, both with respect to concentrations of 5-methyltetrahydrofolate, for the presence of cerebral folate deficiency, and also with respect to abnormal cytokine or chemokine levels, for the presence of immune abnormalities. Neither of these explorations yielded results that indicated consistent abnormalities of these types were apparent in this sample. With respect to cytokines and chemokines, some modulators of immune function (e.g., EGF and soluble CD40 ligand) were increased in the autism group; however, no evidence of group differences in traditional markers of active inflammation (e.g., IL-6, TNF, IL-1) were observed in the serum. Further, within-subject stability (measured by estimated intraclass correlations) of most analytes was low, indicating that a single measurement is not a reliable prospective indicator of concentration for most analytes. Additionally, in participants with autism, there was little correspondence between the blood and CSF profiles of cytokines, chemokines, and growth factors, suggesting that peripheral markers may not optimally reflect the immune status of the central nervous system. We also analyzed data from MRI scans, which found signals of abnormal myelination in specific brain areas (i.e. corpus callosum) in children with autism, as well as other indicators of abnormalities in brain white matter and a relative thinner cortex in specific key regions in the brain. Evidence of previously reported abnormalities of early brain growth was also investigated, both through imaging as well as head circumference records, with data indicating previous findings may have spuriously overestimated increased head size in autism due to inadequate norms. Longitudinal data found a lack of typical age-related cortical thickness decreases in children with autism, and increases in cortical thickness in some areas were actually associated with language gains. As expected, genetic abnormalities were found in approximately 10% of the subjects and the aberrations are being evaluated for clinical significance. Further, whole exome sequencing of the cohort is currently under analysis, including the relationship of sequencing data and phenotypic variations. Other phenotyping studies have been recently completed. Each of them shares the goal of providing a deeper understanding of the autism phenotype and its relationship to etiology, clinical course and outcome. One of these projects is a prospective, longitudinal investigation of toddlers considered to be at-risk for ASD because of the presence of early language delays (Protocol NCT01339767). This study aimed to delineate early communicative impairments that predict ASD and to distinguish these from non-specific markers of (non-autistic) developmental delays. Data collection has now been completed, and analyses will also examine the relationship of communication impairments to abnormalities of brain structure and function assessed by electroencephalograms (EEGs), structural MRIs, and functional MRI scans, with the goal of identifying specific risk and resilience factors for ASD. This study included a pilot of near-infrared spectroscopy (NIRS), with findings indicating lower cognitive (including language) abilities in toddlers were associated with fNIRS measures of both lower oxygenation variability and more extreme lateralization index values (which indicate differences in oxy-hemoglobin saturation from left and right prefrontal cortex). We also finished data collection for a NIRS study of children aged 4 to 8 years of age during this time period. The goal here was to pilot this relatively new form of functional neuroimaging in children who are too young or behaviorally-challenged to tolerate functional MRI scanning procedures -- a key group that is missing from much of the neuroimaging literature on autism. Using executive functioning tasks, this study obtained reliable and informative brain activation data on children with autism as young as 4 years old as well as typically developing controls. In addition to finding developmentally sensitive parameters of cerebral autoregulation, we also analyzed prefrontal activation in typically developing children during two executive functioning tasks and found that the strength of activation increased with age and with improvements in task performance. A second NIH Bench-to-Bedside award is now being used to investigate typically developing and at risk infants, to explore use of a combined fNIRS and EEG system (see: http://clinicalcenter.nih.gov/ccc/btb/awards1.html Mirror neuron network dysfunction as an early biomarker of neurodevelopment).